Cellular accumulation and cytotoxic effects of zinc oxide nanoparticles in microalga Haematococcus pluvialis

Introduction

Zinc oxide nanoparticle (ZnO NP) has been widely used in ceramics, rubber, glass, cement, plastics, lubricants, pigments, paints, skincare, pharmaceutical, food, and textiles industries due to its catalytic, antimicrobial, anti-corrosion, anti-aging, anti-friction, anti-ultraviolet light, and deodorizing properties with large surface for the reactions (Yung et al., 2015; Hou et al., 2018; Moezzi, Mcdonagh & Cortie, 2012; Klingshirn, 2007; Piccinno et al., 2012; Popov et al., 2005). ZnO NP is a transparent and conductive metal oxide nanoparticle that provides clear coatings on transparent surfaces, and at the same time inherits the property of piezoelectricity, which makes it attractive for the manufacturing of electronic products (Ma, Williams & Diamond, 2013). Recently, ZnO NPs have been used in numerous chemotherapeutic drugs such as doxorubicin, 5-flurouracil, and doxorubicin as a carrier (Babu et al., 2017). The estimated global annual production of ZnO NPs as of 2010 was >30,000 metric tons (Keller et al., 2013). The extensive usage of ZnO NPs increases the release of this nanoparticle into the environment and may potentially bring adverse effects to the ecosystem and human health (Ma, Williams & Diamond, 2013). Hence, it is essential to investigate the impact of ZnO NPs on the environment, especially on the aquatic organisms. Previous studies revealed the potential of microalgae as model organisms for the toxicity study of metallic oxide NPs in the environment (Franklin et al., 2007; Griffitt et al., 2008; Navarro et al., 2008; Battin et al., 2009; Manzo et al., 2013; Djearamane et al., 2018). Microalgae have been used as a bio-indicator for freshwater pollutants due to their high bioaccumulation capabilities (Barhoumi & Dewez, 2013) and the ability to indicate the amount of pollutants in water through the changes in algal biomass and photosynthetic pigments (Zhou et al., 2014). The responses of algae to chemical pollutants might be species-specific, thus it is important to conduct tests on different algal species to confirm their responses to certain pollutants (Li et al., 2017).

Haematococcus pluvialis is a kind of unicellular, motile, biflagellate, green, and freshwater microalga, with the size of 20–50 μm in diameter and 8–12 μm long (Shah et al., 2016; Kang et al., 2005). H. pluvialis grows in freshwater bodies (Burchardt et al., 2006; Klochkova et al., 2013; Chekanov et al., 2014) and it consists of carotenoids (>1.75%), astaxanthin (>1.5%), fatty acids (7–25%), proteins (20–30%), carbohydrates (30–40%), and minerals. It is considered as the best natural source of the commercial product astaxanthin, which is a well-known antioxidant, anticancer, and anti-inflammatory substance (Honga, Choib & Simb, 2016; Matos et al., 2017). In addition, H. pluvialis has been used as diets for farmed salmon, trout, sea bream, prawns, and ornamental fish (Dore & Cysewski, 2003). H. pluvialis cells are very sensitive to environmental stress and they undergo morphological alterations under various environmental conditions (Hata et al., 2001; Imamoglu, Sukan & Dalay, 2007). Lone et al. (2013) reported that the presence of metallic NPs in the aquatic environments due to several anthropogenic activities might cause detrimental effects on the nutritional quality of the nutrient microalgae through biochemical and physiological alterations. Comotto et al. (2014) revealed the sensitivity of H. Pluvialis towards titanium dioxide nanoparticles (TiO₂ NPs) through a reduction in algal biomass. Hence, the additional information regarding the toxicity effects of ZnO NPs on the nutrient microalga H. pluvialis will be useful for the environmental impact assessment of ZnO NPs in the aquatic environment. The information will also help to design methods for screening the contamination of ZnO NPs in microalgae. Otherwise, the intake of ZnO NPs contaminated microalgae may cause health hazards to the consumers. In this paper, cellular accumulation and the corresponding cytotoxic effects of ZnO NPs on freshwater microalga H. pluvialis are reported.

Materials and Methods

Results

Characterization of ZnO NPs

Scanning electron microscopy micrograph of ZnO nanopowder showed NPs of varied sizes and shapes, such as the sphere, quasi-sphere, hexagon, and rod with agglomerates. The size of the particles was measured to be 51.8 nm with a range of 46.7–56.4 nm (Fig. 1A) and the EDX spectrum of ZnO nanopowder (Fig. 1B) presented peaks corresponded to zinc and oxygen molecules that confirmed the elemental composition of ZnO NPs. Further, the XRD spectrum of the nanopowder (Fig. 1C) displayed strongest diffraction peaks at 31.7°, 34.36°, and 36.19° and also the diffraction peaks at 47.05°, 56.09°, 62.38°, 65.90°, 67.45°, and 68.60°. All diffraction peaks of nanopowder corresponded to the characteristic hexagonal wurtzite crystalline structure of ZnO NPs (Ramesh, Anbuvannan & Viruthagiri, 2015).

Qualitative analysis of ZnO NPs accumulation in algal biomass

Scanning electron microscopy image of control algal cells showed smooth spherical non-aggregated cells (Fig. 2A) with no noticeable peak for zinc in the EDX spectrum (Fig. 2B). Whereas, the SEM micrograph of the algal cells treated with ZnO NPs displayed surface accumulation of NPs and the resultant algal cell aggregation (Fig. 2C). The EDX spectral peaks characteristic for zinc were noticed in the algal biomass treated with ZnO NPs (Fig. 2D) that confirmed the accumulation of ZnO NPs in algal biomass.

Quantification of ZnO NPs accumulation in algal cells

The present study exhibited a dose- and time-dependent accumulation of ZnO NPs in algal cells. The ICP OES results showed a significant (p < 0.05) accumulation of zinc in algal cells from 24 h onward for all the tested concentrations of ZnO NPs (10–200 μg/mL). The maximum accumulation of zinc in the algal cells was reported at 96 h with the resultant values of 6.27 ± 0.34, 9.97 ± 0.8, 13.3 ± 0.9, 15.36 ± 1.2, and 18.36 ± 1.38 pg/cell (Fig. 3A) at 10, 50, 100, 150, and 200 mg/L of ZnO NPs, respectively.

Growth inhibitory effect of ZnO NPs on H. pluvialis

The present study results the growth inhibition of ZnO NPs on H. pluvialis reflected by the loss in cell viability, algal biomass, and photosynthetic pigments. The treatment of ZnO NPs caused significant (p < 0.05) reduction in cell viability and biomass of H. pluvialis from 72 h for all the tested concentrations of ZnO NPs (10–200 μg/mL). The highest growth inhibition was observed at 96 h with 52.78 ± 5.12% loss in cell viability (Fig. 3B) and 49.35 ± 3.69% reduction in algal biomass (Fig. 3C) for 200 μg/mL of ZnO NPs, as compared to control. The growth inhibition of ZnO NPs on H. pluvialis was further investigated by measuring the loss in photosynthetic pigments. The results show that the exposure to 10–200 μg/mL ZnO NPs lead to significant loss (p < 0.05) in chlorophyll-a at 24 h and carotenoids at 48 h. The maximum reduction in chlorophyll-a and carotenoids were reported to be 63.27 ± 2.4% (Fig. 4A) and 43.35 ± 3.57% (Fig. 4B) respectively, at 200 μg/mL of ZnO NPs for the exposure duration of 96 h. Similarly, the toxicity of ZnO NPs instigated a significant (p < 0.05) decline in astaxanthin content from 48 h at 10–200 μg/mL. The maximum reduction in astaxanthin was reported at 96 h with the resultant value of 47.91 ± 3.12% for 200 μg/mL of ZnO NPs (Fig. 4C). The results of the study demonstrated a characteristic time- and dose-dependent growth inhibitory effect of ZnO NPs on H. pluvialis through a progressive decrease in cell viability, algal biomass, and the photosynthetic pigments.

Morphological examination of algal cells treated with ZnO NPs

The light microscopic image of algal cells without ZnO NPs treatment (control) showed spherical, membrane-intact cells with numerous motile cells (Fig. 5A). Whereas, the treatment of NPs resulted in adsorption and aggregation of NP agglomerates on algal cells (Fig. 5B), algal cells aggregation, degradation, and bleaching of cells by loss of chlorophyll through cell wall rupture (Fig. 5C), aggregation of algal cells with many ghost cells (Fig. 5D), and clustering of distorted cells (Fig. 5E). Similarly, the SEM image of control cells displayed smooth spherical cells with intact cell membranes (Fig. 6A). On the contrary, ZnO NPs treated algal cells showed entrapment of cells with NP agglomerates (Fig. 6B), algal cells aggregation (Fig. 6C), cell distortion with altered cell membrane (Fig. 6D), cell membrane rupture and the subsequent cell rupture (Fig. 6E), and aggregates of distorted cells (Fig. 6F).

Furthermore, the TEM micrograph of the control cells shown in Fig. 7A, revealed a thick cell wall surrounding the cell and highly developed chloroplasts with densely packed thylakoids in the cytoplasm. Figure 7C shows the control cell displaying lipid vesicles in the cytoplasm with astaxanthin accumulation. Whereas, the cells treated with ZnO NPs showed complete degradation of thick cell wall together with loss of smooth layers (Fig. 7B). ZnO NPs treatment resulted in plasmolysis with leakage of cytoplasmic contents, deformation of lipid vesicles, and degradation cytoplasmic organelles (Fig. 7D).

Discussion

Gupta, Sharma & Singh (2014) described two mechanisms in the uptake of metal ions into the algal cells. Firstly, the adsorption of metal ions on the algal surface and the subsequent entry into the cells through cell membranes to get internalized into the cytoplasmic organelles. According to the mechanisms described by Gupta, Sharma & Singh (2014), the results of the present study determined the accumulation of ZnO NPs in the algal biomass through SEM EDX (Figs. 2C and 2D). Similar results were reported by Dmytryk, Saeid & Chojnacka (2014), Zinicovscaia et al. (2016), and Djearamane et al. (2018) who demonstrated the cellular accumulation of selenium, zinc and ZnO NPs in the algal biomass of Spirulina platensis through EDX analysis. Furthermore, the quantification of ZnO NPs accumulated in algal cells using ICP OES exhibited a dose- and time-dependent increase in cellular accumulation of ZnO NPs in H. pluvialis with highest zinc accumulation of 6.27–18.36 pg/cell (Fig. 3A) for 10–200 mg/L of ZnO NPs at 96 h. Gunawan et al. (2013) demonstrated a dose-dependent cellular uptake of zinc in green alga Chlamydomonas reinhardtii with the cellular uptake of zinc ranged from five fg/cell at one mg/L to 18 fg/cell at 100 mg/L on day 8 when exposed to the increasing concentrations of ZnO NPs from one to 100 mg/L. In addition to that, the exposure of duckweed Spirodela polyrhiza to 1, 10, and 50 mg/L of ZnO NPs resulted in dose-dependent cellular accumulation of 2.8, 3.6, and 4.5 mg of zinc/g dry weight on day 4 (Hu et al., 2013). A study by Liu et al. (2019) demonstrated the accumulation of silver NPs (Ag NPs) coated with sodium citrate with particle size 20 nm (CIT20) and polyvinylpyrrolidone with particle size 20 nm (PVP20) in zebrafish with the reported values of 30.3 and 66.2 mg/g of silver in intestines, when exposed to 0.61 mg/L of CIT20 and 0.67 mg/L of PVP20 at 96 h respectively. Shen et al. (2013) reported the higher accumulation of zinc in macrophages with 13, 24.6, 99.4 pg/cell compared to monocytes with accumulated zinc of 0.3, 5.1, 22.9 pg/cell at 24 h when treated with 10, 50, and 100 mg/L of ZnO NPs respectively. Similar to the study findings of Shen et al. (2013), the current results revealed a strong relationship between the cellular accumulation of ZnO NPs and cell mortality.

The cytotoxicity parameters of the present study evidently demonstrated a characteristic dose- and time-dependent loss in cell viability and a decrease in algal biomass which confirmed the growth inhibitory effect of ZnO NPs on H. pluvialis. A similar trend was observed with the study conducted on freshwater microalga Chlorella vulgaris (Suman, Rajasree & Kirubagaran, 2015) and the marine microalga S. platensis (Djearamane et al., 2018). However, the present study results reported lower sensitivity of H. pluvialis towards ZnO NPs toxicity in comparing with S. platensis and C. vulgaris. Nevertheless, H. pluvialis showed a higher sensitivity to the toxic effects of ZnO NPs when compared with marine alga S. polyrhiza (Hu et al., 2013). A study by Yung et al. (2017) confirmed that ZnO NPs had a higher toxicity effect than ZnO and ZnSO₄ on the marine diatom Thalassiosira pseudonana. Numerous earlier studies have reported the sensitivity of H. pluvialis to the adverse effects of metals, metallic NPs, and environmental stress. Exposure of H. pluvialis to different concentrations of NaCl salt (0.25–2% w/v) for 4 days resulted in 56.6% loss in cell viability and also triggered subsequent steep fall of biomass together with the microscopic evidence of cell lysis at the highest concentration of NaCl (Sarada, Tripathi & Ravishankar, 2002). Li et al. (2008) reported 8.4%, 13.1%, and 19.7% loss in cell viability of H. pluvialis at 48 h under various stress conditions when treated with ferrous sulfate (FS), FS + high intensity light (HL) (200 μmol m⁻² s⁻¹) and FS + HL + sodium acetate respectively. A recent investigation by Comotto et al. (2014) reported an 18.1% decrease in biomass of H. pluvialis on day 9 with the treatment of 100 μg/mL of TiO₂ NPs.

In addition, the current results demonstrated a dose- and time-dependent reduction in photosynthetic pigments of H. pluvialis corresponding to the loss in cell viability and algal biomass due to the treatment with ZnO NPs. The highest fall in chlorophyll-a, carotenoids, and astaxanthin of H. pluvialis were observed at 200 μg/mL on day 4 with the reported values of 63.27%, 43.35%, and 47.91%, respectively (Fig. 4). A similar result was reported by Vidhyavathi et al. (2008) where the stress induced by salt caused 90% and 56.68% reduction in chlorophyll and astaxanthin with a 54.68% decrease in carotenoids of H. pluvialis when exposed to 17.1 mM of NaCl on day 9. Besides the salt stress, exposure of H. pluvialis to the high intensity of light (97 μmol m⁻²s⁻¹) for 48 h resulted in 14.9% and 8.45% decrease in chlorophyll and total carotenoids, respectively (Vidhyavathi, Sarada & Ravishankar, 2009). Numerous studies have described the sensitivity of microalgal pigments toward the toxicity of metals and metallic NPs. Sadiq et al. (2011) showed a dose- and time-dependent loss in the chlorophyll of microalgae Chlorella sp. and Scenedesmus sp. upon treating with alumina NPs for 72 h. Besides, Oukarroum et al. (2012) demonstrated a concentration-dependent reduction in the chlorophyll of C. vulgaris and Dunaliella tertiolecta when treated with Ag NPs for 24 h. Sadiq et al. (2011), Djearamane et al. (2019a), (2019b), and Barhoumi & Dewez (2013) have reported the strongest toxic effects of metallic oxide NPs on the electron transport chain of photosynthesis in C. vulgaris that resulted in a dose-dependent reduction in the photosynthetic pigment. Unfavorable growth conditions to microalgae such as the stress caused by salt, high light intensity, chemicals, metals, and metal NPs cause reduction in chlorophyll content of microalgae and that could be the major reason for algal growth inhibition as the chlorophyll plays a key role in photosynthesis (Gupta, Sharma & Singh, 2014). Treatment of metals to microalgae results in the destruction of thylakoids that causes a reduction in photosynthetic pigments, which in turn severely affects the photosynthetic activity and causes growth inhibition or cell death (Arunakumara, Zhang & Song, 2008).

Furthermore, the light microscopic images of H. pluvialis treated with ZnO NPs revealed wrapping of algal cells with NPs and presence of ghost cells (Fig. 5), in addition to, adsorption of NP agglomerates on algal cells, cell aggregation, alteration in cell structure with wrinkled surface, cell membrane rupture, cell distortion, and cell rupture shown by scanning electron microscopic images (Fig. 6). In accordance with our findings, Comotto et al. (2014) observed the cell aggregation and cell wall degradation in C. vulgaris, H. pluvialis, and S. platensis upon treating with TiO₂ NPs. Dong et al. (2014) showed strongly wrinkled and damaged cell walls in hydrochloric acid-acetone treated H. pluvialis cells. Harker, Tsavalos & Young (1996) demonstrated the fully bleached ghost cells of H. pluvialis containing no chlorophyll or carotenoid when exposed to high salt and high light intensity. Such bleaching of cells is thought to happen due to the metabolic imbalance caused by environmental stress and also may be due to the cytoplasmic leakage of cells through cell membrane rupture. In addition, TEM micrographs of ZnO NPs treated H. pluvialis showed complete loss of thick cell wall, plasmolysis, destruction of photosynthetic apparatus, degradation and leakage of cytoplasmic contents (Fig. 7). Similar findings were reported by Kasemets et al. (2009) who demonstrated irregularly shaped cells such as shrunken and deformed cells with crushed cell wall and cytoplasmic leakage in yeast Saccharomyces cerevisiae upon treating with ZnO NPs. Lee et al. (2013) exhibited phytotoxic effects of ZnO NPs on the medicinal plant Fagopyrum esculentum through reduction in biomass of seedling along with damage to surface of root by the aggregates of NPs. Most recently, Hou et al. (2019) demonstrated the genotoxic effects of ZnO NP on zebrafish that the treatment of ZnO NPs affected DNA replication at different phases of cell division which resulted in growth inhibition of zebrafish.

The aggregation and adsorption of NPs on algal surface occur due to the large surface area of NPs and also because of the electrostatic attraction of positively charged ZnO NPs with negatively charged functional groups present in the algal cell wall. The accumulation of nanosized particles on algal cell surface alters cell membrane permeability and causes cell wall damage, that enables excessive entry of NPs into the cells and brings disturbance in the vital functions of internal organelles which subsequently leading to cell death (Kumar et al., 2011; Bhuvaneshwari et al., 2015). Similarly, the algal growth inhibition results from the penetration of NPs into the cell envelope and the following disruption in cell membranes causing cell membrane rupture and leakage of intracellular contents (Chen et al., 2012). The surface binding and the consequent accumulation of nanosized particles on the algal cell surface compromise the cell membrane integrity and cell morphology, and eventually resulting in cell death due to the mechanical damage (Djearamane et al., 2018) or by the intracellular dissociation of metal ions from the internalized NPs that disturbs cellular metabolism (Lin & Xing, 2008). Further, the entrapment of large aggregates of NPs on algal cells reduces the light availability to algal cells (Hazeem et al., 2016) and also the adsorption of nutrients by NPs impairs the availability of nutrients to algal cells, ultimately resulting in growth inhibition (Tang et al., 2013).

Conclusion

In a nutshell, the treatment of ZnO NPs on H. pluvialis resulted in a characteristic dose- and time-dependent accumulation of ZnO NPs in algal cells and triggered the growth inhibitory effects on algal cells together with significant surface and intracellular damages. In addition, the present study demonstrated the following mechanisms that are believed to be responsible for the cytotoxicity effects of ZnO NPs on algal cells; entrapment of algal cells with NPs agglomerates that caused cell membrane damage; physical adsorption and the subsequent uptake of NPs into the algal cells resulted in the destruction of intracellular organelles including photosynthetic apparatus.

Supplemental Information